They have been tested different forms of colorectal carcinoma treatment, however up to day the surgery it has been the only curative way. The surgery has allowed reaching higher percents of survival, when the detection of the tumour is in an early stage, but unfortunately the most cases are diagnosticated when the tumour has metastized.
In this moment, the strategy to increase survival includes the diagnosis, the therapeutic and epidemiology, in stages wherein it has not been produced the dissemination of the disease to external layers of the organs and the tumour is still surgically curable. In the way, the knowledge of epidemiological factors as well as the development of new therapeutically methods will help to increase the survival.
The use of monoclonal antibodies (Mabs) or their fragments, labelled with radioactive isotopes for the detection of cancer through immunogammagraphic methods, has been used in the last years. The Mabs have shown potential to be used as carriers of radioisotopes and to be targeted to the associated tumour antigens.
Some of the radiolabelled antibodies have been used to detect tumours associated with carcinoembrionary antigens (CEA). The antibodies against CEA, labelled with I-131 or I-125 are used to detect tumours producing CEA or associated with this marker (U.S. Pat. No. 3,663,684, U.S. Pat. No. 3,867,363 y U.S. Pat. No. 3,927,193). Also, Mabs can be labelled with Tc-99m to get molecules for “in vivo” diagnosis.
The development of the hybridoma antibody technique by Köhler and Milstein revolutionised the discipline of immunochemistry and provided a new family of reagents with potential applications in the research and clinical diagnosis of diseases (Köhler G; Milstein C. (1975) Nature 256, 495-497). These antibodies have not shown strong therapeutic efficacy, while it has become routine to produce mouse monoclonal antibodies (mAbs) for use in basic research and clinical diagnosis, it has been difficult to use these for “in vivo” immunotherapy, because they have reduced half life in humans, poor recognition of mouse antibodies effector domains by the human immune system and also because foreign immunoglobulins can elicit an antiglobulin response (HAMA response) that may interfere with therapy.
The development of the genetic engineering has revolutionised the ability to genetically manipulate antibody genes and then to produce mAbs having decreased or eliminated antigenicity and enhanced desired effector functions, when these antibodies are used in the treatment or diagnosis of some pathologies. These manipulations have provided an alternative where a murine mAb can be converted to a predominantly human form with the same antigen binding properties (Morrison S. L; et al 1984, P.N.A.S. USA, 81,6851-6855).
Recently they have been developed some methods in order to humanise murine or rat antibodies and decrease xenogenic response against foreign proteins when they are used in humans.
One of the first intents to reduce antigenicity, has been by producing “chimeric” antibodies. In these molecules, the variable domains were inserted into human frameworks, in this way not only it can be reached the decrease of the immunogenicity but also the improvement of effector functions, because they are humans and therefore recognised by the immune system (Morrison S. L et al (1984) P.N.A.S, USA 81, 6851-6855). These chimeric molecules retain the recognition of the original antigen and its constant region is not immunogenic, although the immunogenicity against murine variable region is retained.
Other authors have attempted to build rodent antigens binding sites directly into human antibodies by transplanting only the antigen binding site, rather than the entire variable domain, from a murine antibody (Jones P. T et al (1986) Nature 321, 522-524, Verhoeyen M et al (1988) Science 239, 1534-1536). They have been developed some applications of this method by Rietchmann (Rietchmann L. et al (1988) Nature 332, 323-327; Quee C. et al (1989) P.N.A.S USA 86,10029-10033), however other authors have worked with reshaped antibodies, which included some murine residues in human FRs in order to recover the affinity for the original antigen (Tempest, P. R (1991) Biotechnology 9, 266-272).
Mateo et al. (U.S. Pat. No. 5,712,120) described a procedure to reduce immunogenicity of the murine antibodies. In this procedure, the modifications are restricted to the variable domains and specifically to the murine frameworks of the chimeric antibodies. Even more, these modifications are only carried out in the FRs regions with amphipatic helix structure, therefore are potential epitopes recognised by T cells. The method proposes to substitute the murine residues inside the amphipatic regions, by the amino acids in the same positions in the human immunoglobulines, of course the amino acids involved in the tridimentional structure of the binding site, it means Verniers zone, canonical structures of the CDRs and the amino acid of the inter-phase between light and heavy chain are excluded.
The antibody modified by the method described by Mateo et al, retains the capacity of the recognition and binding to the antigen, that recognised the original antibody and it results less immunogenic because of this it is got an increase of the therapeutic efficacy. Through this procedure only few mutations are necessary to obtain modified antibodies that shown reduced immunogenicity compared with chimeric antibodies.
The IOR C5 murine monoclonal antibody (patent application WO 97/33916) is an IgG1 isotype, obtained from immunisation of Balb/c with SW1116 cells (colorectal adenocarcinoma), recognised an antigen expressed preferentially in the surface and cytoplasm of the malignant and normal colorectal cells. This antibody does not recognise neither CEA, Lewis a, Lewis b, asialylated Lewis, membranes of normal mononuclear cells antigens nor red globules (Vazquez A. M. et al, Hybridoma 11, pag. 245-256, 1992).
Western blotting studies using SW1116 membranes extract showed that this antibody recognized a glycoprotein complex which was denominated ior C2, with two molecular weight forms (145 and 190 Kda) (Vázquez A. M. et al, Year Immunol. Basel, Karger, vol. 7, pag. 137-145,1993).
Also it is known from the state of the art that using genetic engineering techniques, recombinant fragments can be constructed from monoclonal antibodies. There are many reports validating the use of different antibody fragments in the “in vivo” diagnosis and the therapeutic of the diseases.
Ira Pastan et al. (EP 0796334 A1) describes the construction of single chain Fv fragments, using variables regions of antibodies that specifically recognised carbohydrates related with Lewis Y antigen. Using these fragments, he developed a method to detect cells bearing this antigen. Also, he gives evidences of the inhibitor effect of these fragments on cells bearing the antigen.